Degradation fragments

ABSTRACT

A pharmaceutical composition comprising a compound of formula (I) wherein X is an electron withdrawing group, Y 1  is hydrogen, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, —SO 2 R 4 , —CO 2 R 4 , —CONHR 4  or —COR 4 , and each of R 1 , R 2  and R 4 , which may be the same or different, is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl or heterocyclyl, or a compound of formula (II) wherein each of Y 2  and Y 3 , which may be the same or different, is hydrogen, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, —SO 2 R 9 , —CO 2 R 9 , —CONHR 9  or —COR 9 , Z is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heterocyclyl, —CH═C(NHR 10 )CH((CH 2 ) m CO 2 R 11 )(C═O)CH 3  or —CH 2 (C═O)CH((CH 2 ) m CO 2 R 11 )(C═O)CH 3 , R 8  is —(CH 2 ),CO 2 R 12 , each of R 5  to R 7  and R 9  to R 12 , which may be the same or different, is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl or heterocyclyl, and each of m and n, which may be the same or different, is 1 to 6 or a compound of formula (III) wherein each of Y 4  to Y 6 , which may be the same or different, is hydrogen, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, —SO 2 R 19 , —CO 2 R 19 , —CONHR 19  or —COR 19 , each of R 16  and R 17 , which may be the same or different, is —(CH 2 ) p CO 2 R 20 , each of R 13  TO R 15  and R 18  to R 20 , which may be the same or different, is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl or heterocyclyl, and p is 1 to 6, or other photolabile degradation product of bilirubin or biliverdin or derivative of a photolabile degradation fragment of bilirubin or biliverdin, or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier or diluent.

[0001] The present invention relates to degradation fragments ofbilirubin or biliverdin, to their derivatives and to their use.

[0002] Erythrocyte lysis (haemolysis) may follow subarachnoidhaemorrhage (SAH), a type of haemorrhagic stroke in which delayedischemic complications is a major factor affecting patient outcome.Haemolysate components such as oxyhaemoglobin, methemoglobin, hemin andbilirubin (BR) are found in cerebral spinal fluid (CSF) from patientswith SAH. SAH induced by subarachnoid injections of lysed blood upregulates expression of haem oxygenase-1 (HO-1), an inducible isoform ofhaem oxygenase, in glia throughout the brain HO-1 catalyses thedegradation of haem resulting in release of biliverdin, CO and free ironand HO-1 expression is a limiting step in the haemoglobin degradationpathway. Biliverdin is subsequently reduced by biliverdin reductase toform BR, a reductant which scavenges reactive oxygen species. Due to itsantioxidant activity, BR can serve as a protective agent in cells,membrane lipids and low density lipoproteins (LDL) exposed to oxidativestress. High levels of BR can be found in subarachnoid clots in theperivascular area and in CSF during SAH (CSF_(SAH)) and SAH inducedcerebral vasospasm.

[0003] Biochemical and clinical studies have indicated a role for oxygenfree radicals in the pathogenesis of vasospasm and neurologicaldysfunction following SAH. Oxyhaemoglobin, a major constituent ofhaemolysate, is a potent generator of reactive oxygen species. Increasesin both enzymatic (arachidonic acid cascade and eicosanoid peroxideproduction) and non-enzymatic (thiobarbituric acid reactive substancesproduction and cholesteryl ester hydroperoxides) lipid peroxidationproducts have been found in models of SAH, suggesting a role foroxidative stress during SAH.

[0004] Cells exposed to oxidative stress have increased activity ofinducible isoform of HO-1 resulting in elevated BR levels. HO-1upregulation is associated with vasious pathological states includingcerebral haemorrhage, cerebral vasospasm following SAH,ischemic-reperfusion and endotoxemia. Although the presence of BR in CSFfrom patients with SAH has been confirmed, BR per se failed to developsignificant arterial spasm in vivo. Therefore, its role in SAH,pathogenesis of vasospasm and complications due to vasospasm hasremained uncertain.

[0005] Generation of reactive oxygen species within the subarachnoidspace following SAH leads to HO-1 upregulation and release of biliverdinand BR, which may serve as targets for reactive oxygen species-mediateddegradation. Under pathological conditions of severe oxidative stress,the oxidative degradation of BR and biliverdin may occur to givecompounds causing vasospasm following SAH.

[0006] The term “vasospasm” as used herein means contraction of bloodvessels particularly in association with the brain.

[0007] The term “vasoconstriction” as used herein means contraction ofthe blood vessels particularly in association with organs other than thebrain.

[0008] The present invention concerns degradation fragments of bilirubinor biliverdin which may be produced under pathological conditions ofsevere oxidative stress, and their derivatives.

[0009] In one aspect the present invention provides a pharmaceuticalcomposition comprising a compound of formula (I)

[0010] wherein X is an electron withdrawing group typically connected tothe ring via an oxygen or carbon atom, such as ═O, ═CH(C═O)R³,═CH(C═O)OR³ or ═CH(C═O)NHR³, Y¹ is hydrogen, alkyl, alkenyl, alkynyl,aryl, heterocyclyl, —SO₂R⁴, —CO₂R⁴, —CONHR⁴ or —COR⁴, and each of R¹ toR⁴, which may be the same or different, is hydrogen, alkyl, cycloalkyl,alkenyl, cycloalkenyl, alkynyl, aryl or heterocyclyl, or

[0011] a compound of formula (II)

[0012] wherein each of Y² and Y³, which may be the same or different, ishydrogen, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, —SO₂R⁹, —CO₂R⁹,—CONHR⁹ or —COR⁹, Z is hydrogen, alkyl, cycloalkyl, alkenyl,cycloalkenyl, alkynyl, aryl, heterocyclyl,—CH═C(NHR¹⁰)CH((CH₂)_(m)CO₂R¹¹)(C═O)CH₃ or—CH₂(C═O)CH((CH₂)_(m)CO₂R¹¹)(C═O)CH₃, R⁸ is —(CH₂)_(n)CO₂R¹², each of R⁵to R⁷ and R⁹ to R¹² which may be the same or different, is hydrogen,alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl or heterocyclyl,and each of m and n, which may be the same or different, is 1 to 6, or

[0013] a compound of formula (III)

[0014] wherein each of Y⁴ to Y⁶, which may be the same or different, ishydrogen, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, —SO₂R¹⁹, —CO₂R¹⁹,—CONHR¹⁹ or —COR¹⁹, each of R¹⁶ and R¹⁷, which may be the same ordifferent, is —(CH₂)_(p)CO₂R²⁰, each of R¹³ to R¹⁵ and R¹⁸ to R²⁰, whichmay be the same or different, is hydrogen, alkyl, cycloalkyl, alkenyl,cycloalkenyl, alkynyl, aryl or heterocyclyl, and p is 1 to 6, or otherphotolabile degradation product of bilirubin or biliverdin or derivativeof a photolabile degradation product of bilirubin or biliverdin, or apharmaceutically acceptable salt thereof, together with apharmaceutically acceptable carrier or diluent.

[0015] It will be appreciated that R¹ to R²⁰, X, Y¹ to Y⁶ and Z can becombinations of the specified groups.

[0016] The term “alkyl” as used herein includes both unsubstituted andsubstituted, straight and branched chain radicals. Typically it is C₁-C₆alkyl, preferably C₁-C₄ alkyl, for example methyl, ethyl, i-propyl,n-propyl, t-butyl, s-butyl or n-butyl. It may also be pentyl, hexyl andthe various branched chain isomers thereof. When the alkyl group issubstituted it typically bears one or more substituents selected fromaryl, cycloalkyl, halogen, trihaloalkyl such as trifluoromethyl,hydroxyl, alkoxyl, aralkoxyl, amino, mono or dialkylamino, nitro, cyano,carbonyl, carboxyl, alkylsulphoxyl or alkylsulphonyl.

[0017] The term “cycloalkyl” as used herein typically means a cycloalkylgroup having 3 to 8 carbons, for example cyclopropyl, cyclopentyl,cyclohexyl and cyclooctyl. A cycloalkyl group may be unsubstituted orsubstituted as the alkyl groups above.

[0018] The term “alkenyl” as used herein includes unsubstituted andsubstituted, straight and branched chain radicals having one or moredouble bonds. Typically it is C₂-C₆ alkenyl such as, for example, allyl,butenyl, butadienyl, pentenyl or hexenyl. When the alkenyl group issubstituted it typically bears one or more substituents as defined abovefor the alkyl groups.

[0019] The term “cycloalkenyl” as used herein typically means acycloalkenyl group having 4 to 8 carbons, for example cyclopentenyl,cyclohexenyl or cyclooctadienyl.

[0020] The term “alkynyl” as used herein includes unsubstituted andsubstituted, straight and branched chain radicals having one or moretriple bonds. Typically it is C₂-C₆ alkynyl, such as butynyl. When thealkynyl group is substituted it typically bears one or more substituentsas defined above for the alkyl groups.

[0021] The term “aryl” as used herein includes both monocyclic andbicyclic aromatic groups which typically contain from 6 to 10 carbons inthe ring portion, such as phenyl or naphthyl. The aryl group isunsubstituted or substituted. When it is substituted the aryl group maybe substituted by, for example, one or more substituents selected fromC₁-C₆ alkyl, C₁-C₆ alkoxyl, trihaloalkyl such as trifluoromethyl,halogen and hydroxyl.

[0022] The term “heterocyclyl” as used herein is typically a 3- to10-membered, saturated or unsaturated heterocyclic ring containing atleast one heteroatom selected from N, O and S and which is optionallyfused to a second 5- or 6-membered, saturated or unsaturatedheterocyclic ring or to an aryl group as defined above. The heterocyclicring may be, for example, pyridine, furan, thiophene, pyrrole,pyrrolidine, pyrimidine, pyrazine, pyridazine, pyrazole or indazole, ora cyclic ether such as glucose. The heterocyclyl group may beunsubstituted or substituted at any position. Suitable substituentsinclude alkyl, for example haloalkyl, aryl, for example phenyl, halogenand hydroxyl.

[0023] The term “halogen” as used herein means fluorine, chlorine,bromine and iodine.

[0024] The term “aralkyl” as used herein refers to alkyl groups aspreviously defined having an aryl substituent, for example benzyl,phenylethyl, diphenylmethyl and triphenylmethyl.

[0025] The term “alkoxyl” or “aralkoxyl” as used herein includes any ofthe above alkyl, cycloalkyl or aralkyl groups linked to an oxygen atom.

[0026] Preferably, X is ═O or ═CH(C═O)NHR³ wherein R³ is hydrogen oralkyl. More preferably, X is ═O or ═CH(C═O)NH₂.

[0027] Preferably, Y¹ is hydrogen.

[0028] Each of R¹ and R² is preferably hydrogen, alkyl or alkenyl. Morepreferably, one of R¹ and R² is hydrogen or alkyl and the other isalkenyl. Still more preferably, one of R¹ and R² is methyl and the otheris —CH═CH₂.

[0029] Preferably, each of Y² and Y³ is hydrogen.

[0030] Z is preferably —CH═C(NHR¹⁰)CH((CH₂)_(m)CO₂R¹¹)(C═O)CH₃ or—CH₂(C═O)CH((CH₂)_(m)CO₂R¹¹)(C═O)CH₃ wherein each of R¹⁰ and R¹¹ ishydrogen or alkyl and m is 1 to 6. More preferably, Z is—CH═C(NHR¹⁰)CH((CH₂)_(m)CO₂R¹¹)(C═O)CH₃ or—CH₂(C═O)CH((CH₂)_(m)CO₂R¹¹)(C═O)CH₃ wherein each of R¹⁰ and R¹¹ ishydrogen or alkyl and m is 1 to 4 Still more preferably, Z is—CH═C(NH₂)CH((CH₂)₂CO₂H)(C═O)CH₃ or —CH₂(C═O)CH((CH₂)₂CO₂H)(C═O)CH₃.

[0031] Each of R⁵ to R⁷ is preferably hydrogen, alkyl or alkenyl. Morepreferably, one of R⁵ and R⁶ is hydrogen or alkyl and the other isalkenyl, and R⁷ is alkyl. Still more preferably, one of R⁵ and R⁶ ismethyl and the other is —CH═CH₂, and R⁷ is methyl.

[0032] Preferably, R⁸ is —(CH₂)₂CO₂R¹² wherein R¹² is hydrogen or alkyl.More preferably, R⁸ is —(CH₂)CO₂H.

[0033] Preferably, each of Y⁴ to Y⁶ is hydrogen.

[0034] Each of R¹³ to R¹⁵ and R¹⁸ is preferably hydrogen, alkyl oralkenyl. More preferably, one of R¹³ and R¹⁴ is hydrogen or alkyl andthe other is alkenyl, and each of R¹⁵ and R¹⁸ is alkyl. Still morepreferably, one of R¹³ and R¹⁴ is methyl and the other is —CH═CH₂, andeach of R¹⁵ and R¹⁸ is methyl.

[0035] Preferably, each of R¹⁶ and R¹⁷ is —(CH₂)₂CO₂R²⁰ wherein R²⁰ ishydrogen or alkyl. More preferably, each of R¹⁶ and R¹⁷ is —(CH₂)₂CO₂H.

[0036] Preferred compositions of the invention are compositions whereinin formula (I) X is ═O or ═CH(C═O)NHR³, Y¹ is as defined above, each ofR¹ and R², which may be the same or different, is hydrogen, alkyl oralkenyl and R³ is hydrogen or alkyl, or

[0037] in formula (II) each of Y² and Y³ is as defined above, Z is—CH═C(NHR¹⁰)CH((CH₂)_(m)CO₂R¹¹)(C═O)CH₃ or—CH₂(C═O)CH((CH₂)_(m)CO₂R¹¹)(C═O)CH₃, each of R⁵ to R⁷ is hydrogen,alkyl or alkenyl, R⁸ is —(CH₂)₂CO₂R¹², each of R¹⁰ to R¹² is hydrogen oralkyl and m is 1 to 6, or

[0038] in formula (III) each of Y⁴ to Y⁶ is as defined above, each ofR¹³ to R¹⁵ and R¹⁸ are hydrogen, alkyl or alkenyl, each of R¹⁶ and R¹⁷is —(CH₂)₂CO₂R²⁰ and R²⁰ is hydrogen or alkyl.

[0039] More preferred compositions of the invention are compositionswherein in formula (I) when X is ═CH(C═O)NH₂, Y¹ is hydrogen, and one ofR¹ and R² is hydrogen or alkyl and the other is alkenyl, or when X is═O, Y¹ is hydrogen, R¹ is alkenyl and R² is hydrogen or alkyl, or

[0040] in formula (II) each of Y² and Y³ is hydrogen, Z is—CH═C(NHR¹⁰)CH((CH₂)_(m)CO₂R¹¹)(C═O)CH₃ or—CH₂(C═O)CH((CH₂)_(m)CO₂R¹¹)(C═O)CH₃, one of R⁵ and R⁶ is hydrogen oralkyl and the other is alkenyl, R⁷ is alkyl, R⁸ is (CH₂)₂CO₂H, each ofR¹⁰ and R¹¹ is hydrogen or alkyl and m is 1 to 4, or

[0041] in formula (III) each if Y⁴ to Y⁶ is hydrogen, one of R¹³ and R¹⁴is hydrogen or alkyl and the other is alkenyl, each of R¹⁵ and R¹⁸ ishydrogen or alkyl and each of R¹⁶ and R¹⁷ is —(CH₂)₂CO₂H.

[0042] Still more preferred compositions of the invention arecompositions wherein in formula (I) when X is ═CH(C═O)NH₂, Y¹ ishydrogen, and one of R¹ and R² is methyl and the other is —CH═CH₂, orwhen X is ═O, Y¹ is hydrogen, R¹ is —CH═CH₂ and R² is methyl, or

[0043] in formula (II) each of Y² and Y³ is hydrogen, Z is—CH═C(NH₂)CH((CH₂)₂CO₂H)(C═O)CH₃ or —CH₂(C═O)CH((CH₂)₂CO₂H)(C═O)CH₃, oneof R⁵ and R⁶ is methyl and the other is —CH═CH₂, R⁷ is methyl and R⁸ is—(CH₂)₂CO₂H or

[0044] in formula (III) each of Y⁴ to Y⁶ is hydrogen, one of R¹³ and R¹⁴is methyl and the other is —CH═CH₂, each of R¹⁵ and R¹⁸ is methyl andeach of R¹⁶ and R¹⁷ is —(CH₂)₂CO₂H.

[0045] As used herein, a pharmaceutically acceptable salt is a salt witha pharmaceutically acceptable acid or base. Pharmaceutically acceptableacids include both inorganic acids such as hydrochloric, sulphuric,phosphoric, diphosphoric, hydrobromic or nitric acid and organic acidssuch as citric, fumaric, maleic, malic, ascorbic, succinic, tartaric,benzoic, acetic, methanesulphonic, ethanesulphonic, benzenesulphonic orp-toluenesulphonic acid. Pharmaceutical acceptable bases include alkalimetal (e.g. sodium or potassium) and alkali earth metal (e.g. calcium ormagnesium) hydroxides and organic bases such as alkyl amines, aralkylamines or heterocyclyl amines.

[0046] The present invention includes all possible isomers of thecompounds of formula (I), (II) or (III), or the other photolabiledegradation fragments of bilirubin or biliverdin, and mixtures thereof,including cis and trans alkene isomers, and diastereomeric mixtures andracemic mixtures, resulting from the possible combinations of (R) and(S) stereochemistry when stereogenic centres are present.

[0047] In another aspect the present invention provides noveldegradation fragments of bilirubin or biliverdin or derivatives ofdegradation fragments of bilirubin or biliverdin. Thus, the presentinvention provides a compound of formula (I), (II) or (III) as definedabove, or other degradation fragment of bilirubin or biliverdin orderivative of a degradation fragment of bilirubin or biliverdin, or asalt thereof, excluding a compound of formula (I) wherein X is ═O, Y¹ ishydrogen, R¹ is —CH═CH₂ and R² is methyl, or a compound of formula (III)wherein each of Y⁴ to Y⁶ is hydrogen, one of R¹³ or R¹⁴ is methyl andthe other is —CH═CH₂, each of R¹⁵ and R¹⁸ is methyl and each of R¹⁶ andR¹⁷ is —(CH₂)₂CO₂H. Suitable salts include those mentioned above asexamples of pharmaceutically acceptable salts.

[0048] The compounds of formula (I), (II) or (III), or the otherdegradation fragments of bilirubin or biliverdin or derivatives ofdegradation fragments of bilirubin or biliverdin, or salts thereof,according to the present invention may be prepared by synthetic methodsknown in the art. They may also be prepared by a process comprisingreaction of bilirubin or biliverdin with an oxidising reagent, forexample oxygen, hydrogen peroxide, potassium permanganate, osmiumtetroxide, chromium (VI) oxide or sodium periodate. Catalysts such astransition metals may be employed in the process. Bilirubin orbiliverdin may suitably be dissolved in an alkali such as sodium orpotassium hydroxide, for example 1 to 5M sodium hydroxide. The solutionmay then be neutralized using, for example, hydrochloric acid such as 5to. 11M HCl, acetic acid, perchloric acid, nitric acid or sulphuricacid. The resulting solution may then be contacted with a free radicalor reactive species. The degradation fragments thus obtained may beextracted, purified and modified, as desired, by methods well known tothose skilled in the art.

[0049] The pharmaceutical compositions of the present invention may beprepared in a conventional way by employing conventional non-toxicpharmaceutical carriers or diluents in a variety of dosage forms andways of administration

[0050] The compositions may, for example, be administered parenterally,either subcutaneously or intravenously or intramuscularly, orintrasternally, or by infusion techniques. The pharmaceuticalcompositions may be in the form of a sterile injectable aqueous oroleaginous suspensions.

[0051] These suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a non-toxic parenterally-acceptable diluent or solvent,for example a solution in 1,3-butanediol. Among the acceptable vehiclesand solvents that may be employed are water, Ringer's solution andisotonic sodium chloride solution. In addition, sterile fixed oils areconventionally employed as a solvent or suspending medium.

[0052] For this purpose any bland fixed oils may be conventionallyemployed including synthetic mono or diglycerides. In addition fattyacids such as oleic acid find use in the preparation of injectables.

[0053] The dose varies according to the activity of the specificcompound, the age, weight, and conditions of the subject to be treated,the type and the severity of the disease, and the frequency and route ofadministration. Typically the dose is from 0.0001 to 50 mg per kg ofbody weight. The amount of active ingredient that may be combined withthe carrier materials to produce a single dosage form will varydepending upon the host treated and the particular mode ofadministration. Dosage unit forms will generally contain between from 5to 500 mg of the active compound.

[0054] The compounds of formula (I), (II) and (III) and the otherphotolabile degradation fragments of bilirubin or biliverdin andderivatives of degradation fragments of bilirubin or biliverdin, andpharmaceutically acceptable salts thereof, have been found to inducevasospasm or vasoconstriction. The compositions according to the presentinvention may therefore find application in treating or inducingvasospasm or vasoconstriction. For example, the compositions of thepresent invention may be used in wound sealing such as peri-operativeclosure of vessels and incisions (either alone or in combination withconventional wound sealing methods), laceration or traumatic woundclosing, or vessel closure following tissue or tumour resection. Thecompositions of the present invention may be in the form of opaqueemulsions or suspensions so that during use degradation of thephotolabile active compound by light is reduced. After surgery, or inthe event that the composition is applied to the wrong site, thephotolabile active compound may be degraded by irradiation of thetreated site.

[0055] The, present invention thus provides a method of treating apatient in need of vasospasm or vasoconstriction, which method comprisesadministering to said patient a non-toxic and therapeutically effectiveamount of a compound of formula (I), (II) or (III), or other photolabiledegradation fragment of bilirubin or biliverdin or derivative of adegradation fragment of bilirubin or biliverdin, or a pharmaceuticallyacceptable salt thereof. The condition of the patient may thereby beameliorated.

[0056] In another aspect the present invention provides a compound offormula (I), (II) or (III), or other photolabile degradation fragment ofbilirubin or biliverdin or derivative of a degradation fragment ofbilirubin or biliverdin, or a pharmaceutically acceptable salt thereof,as defined above for use in a method of treatment of the human or animalbody.

[0057] In another aspect the present invention provides use of acompound of formula (I), (II) or (III), or other photolabile degradationfragment of bilirubin or biliverdin or derivative of a degradationfragment of bilirubin or biliverdin, or a pharmaceutically acceptablesalt thereof, as defined above in the manufacture of a medicament foruse in treating or inducing vasospasm or vasoconstriction.

[0058] In another aspect the present invention provides a diagnosticcomposition comprising a compound of formula (I) as defined abovewherein when X is ═CH(C═O)NH₂, Y¹ is hydrogen, and one of R¹ and R² ismethyl and the other is —CH═CH₂, or when X is ═O, Y¹ is hydrogen, R¹ is—CH═CH₂ and R² is methyl, or

[0059] a compound of formula (II) as defined above wherein each of Y²and Y³ is hydrogen, Z is —CH₂(C═O)CH((CH₂)₂CO₂H)(C═O)CH₃, one of R⁵ andR⁶ is methyl and the other is —CH═CH₂, R⁷ is methyl, R⁸ is (CH₂)₂CO₂H,or

[0060] a compound of formula (III) as defined above wherein one of R¹³and R¹⁴ is methyl and the other is —CH═CH₂, each of R¹⁵ and R¹⁸ ismethyl and each of R¹⁶ and R¹⁷ is —(CH₂)₂CO₂H, or other degradationfragment of bilirubin or biliverdin, or a salt thereof, and a diluent orcarrier. Suitable salts include those mentioned above as examples ofpharmaceutically acceptable salts.

[0061] In one embodiment the diagnostic composition comprises aphotolabile degradation fragment of bilirubin or biliverdin.

[0062] In another aspect the present invention provides a method fordiagnosing vasospasm or vasoconstriction in a host comprisingdetermining the presence or absence of a compound of formula (I) asdefined above wherein when X is ═CH(C═O)NH₂, Y¹ is hydrogen, and one ofR¹ and R² is methyl and the other is —CH═CH₂, or when X is ═O, Y¹ ishydrogen, R¹ is —CH═CH₂ and R² is methyl, or a compound of formula (II)as defined above wherein each of Y² and Y³ is hydrogen, Z is—CH₂(C═O)CH((CH₂)₂CO₂H)(C═O)CH₃, one of R⁵ and R⁶ is methyl and theother is —CH═CH₂, R⁷ is methyl, R⁸ is (CH₂)₂CO₂H, or

[0063] a compound of formula (III) as defined above wherein one of R¹³and R¹⁴ is methyl and the other is —CH═CH₂, each of R¹⁵ and R¹⁸ ismethyl and each of R¹⁶ and R¹⁷ is —(CH₂)₂CO₂H, or other degradationfragment of bilirubin or biliverdin, or a salt thereof, wherein thepresence of the compound of formula (I), (II) or (III), or thedegradation fragment of bilirubin or biliverdin, or salt thereof,indicates that the host has vasospasm or vasoconstriction.

[0064] In one embodiment the method comprises

[0065] (a) contacting a sample from the host with an agent that binds tothe compound of formula (I), (II) or (III), or the other degradationfragment of bilirubin or biliverdin, or salt thereof, and

[0066] (b) detecting whether the agent binds to components in thesample, thereby determining the presence or absence of the compound offormula (I), (II) or (III), or the other degradation fragment ofbilirubin or biliverdin, or salt thereof.

[0067] The agent may be any agent capable of binding to the compound offormula (I), (II) or (III), or the other degradation fragment ofbilirubin or biliverdin, or salt thereof, for example an antibody or alabelled antibody.

[0068] An antibody to the compound of formula (I), (II) or (III), or theother degradation fragment of bilirubin or biliverdin, or salt thereof,may be produced by raising antibody in a host animal against the wholeor part of the compound of formula (I), (II) or (III), or the otherdegradation fragment of bilirubin or biliverdin, or salt thereof(hereinafter “the immunogen”). Methods of producing monoclonal andpolyclonal antibodies are well-known. A method for producing apolyclonal antibody comprises immunising a suitable host animal, forexample an experimental animal, with the immunogen and isolatingimmunoglobulins from the serum. The animal may therefore be inoculatedwith the immunogen, blood subsequently removed from the animal and theIgG fraction purified. A method for producing a monoclonal antibodycomprises immortalising cells which produce the desired antibody.Hybridoma cells may be produced by fusing spleen cells from aninoculated experimental animal with tumour cells (Kohler and Milstein,Nature 256, 495-497, 1975).

[0069] An immortalized cell producing the desired antibody may beselected by a conventional procedure. The hybridomas may be grown inculture or injected intraperitoneally for formation of ascites fluid orinto the blood stream of an allogenic host or immunocompromised host.Human antibody may be prepared by in vitro immunisation of humanlymphocytes, followed by transformation of the lymphocytes withEpstein-Barr virus.

[0070] For the production of both monoclonal and polyclonal antibodies,the experimental animal is suitably a goat, rabbit, rat or mouse. Ifdesired, the immunogen may be administered as a conjugate in which theimmunogen is coupled to a suitable carrier. The carrier molecule istypically a physiologically acceptable carrier. The antibody obtainedmay be isolated and, if desired, purified.

[0071] The sample used in the method for diagnosing vasospasm orvasoconstriction according to the present invention may be any suitablesample from human or animal. The sample is typically a cerebral spinalfluid or blood sample. The sample may be processed before it is used inthe method, for example it may be diluted, typically in water, saline orsaline containing a buffer (any of these diluents may additionallycomprise detergent).

[0072] An antibody used in the method of the invention may either be awhole antibody or a fragment thereof which is capable of binding thecompound of formula (I), (II) or (III), or the other degradationfragment of bilirubin or biliverdin, or salt thereof. Typically theantibody is monoclonal. Such a whole antibody is typically an antibodywhich is produced by any of the methods of producing an antibody whichare discussed herein. Typically the antibody is a mammalian antibody,such as a primate, human, rodent (e.g. mouse or rat), rabbit, ovine,porcine, equine or camel antibody. The antibody can be any class orisotype of antibody, for example IgM, but is preferably IgG.

[0073] The fragment of whole antibody that can be used in the methodcomprises an antigen binding site, e.g. Fab or F(ab)₂ fragments. Thewhole antibody or fragment may be associated with other moieties, suchas linkers which may be used to join together 2 or more fragments orantibodies. Such linkers may be chemical linkers or can be present inthe form of a fusion protein with the fragment or whole antibody. Thelinkers may thus be used to join together whole antibodies or fragmentswhich have the same or different binding specificities, e.g. that canbind the same or different compounds of formula (I), (II) or (III), orother degradation fragments of bilirubin or biliverdin, or saltsthereof. The antibody may be a bispecific antibody which is able to bindto two different antigens (or antigenic surfaces), typically any two ofthe compounds of formula (I), (II) or (III), or other degradationfragments of bilirubin or biliverdin, or salts thereof, mentionedherein. The antibody may be a ‘diabody’ formed by joining two variabledomains back to back. In one embodiment the antibody is a chimericantibody comprising sequence from different natural antibodies, forexample a humanised antibody.

[0074] Generally the method is carried out in an aqueous solution. Thesample and/or the antibody may be present is solution in the method. Inparticular embodiments (some of which are discussed below) the agent orsample is immobilised on a solid support. Typically such a support isthe surface of the container in which the method is being carried out,such as the surface of a well of a microtitre plate.

[0075] In the method, determining whether the agent binds a compound offormula (I), (II) or (III), or other degradation fragment of bilirubinor biliverdin, or sat thereof, in the sample may be performed any methodknown in the art for detecting binding between two moieties. The bindingmay be determined by measurement of a characteristic in either theantibody or the compound of formula (I), (II) or (III), or the otherdegradation fragment of bilirubin or biliverdin, or salt thereof, thatchanges when binding occurs, such as a spectroscopic change.

[0076] In a preferred embodiment the agent is immobilised on a solidsupport (such as the supports discussed above). When the sample iscontacted with the agent compounds of formula (I), (II) or (III), orother degradation fragments of bilirubin or biliverdin, or saltsthereof, bind to the agent. Optionally the surface of the solid supportis then washed to remove any compound from the sample which is not boundto agent. The presence of the compound of formula (I), (II) or (III), orthe other degradation fragment of bilirubin or biliverdin, or saltthereof, bound to the solid support (through the binding with the agent)can then be determined, indicating that the compound of formula (I),(II) or (III), or the other degradation fragment of bilirubin orbiliverdin, or salt thereof, is bound to the agent. This can be done forexample by contacting the solid support (which may or may not have thecompound of formula (I), (II) or (III), or the other degradationfragment of bilirubin or biliverdin, or salt thereof, bound it) with asubstance that binds the compound of formula (I), (II) or (III), or theother degradation fragment of bilirubin or biliverdin, or salt thereof,specifically. This agent may be labelled either directly or indirectlyby a detectable label.

[0077] Typically the substance is a second agent which is capable ofbinding the compound of formula (I), (II) or (III), or the otherdegradation fragment of bilirubin or biliverdin, or salt thereof, in aspecific manner whilst the compound of formula (I), (II) or (III), orthe other degradation fragment of bilirubin or biliverdin, or saltthereof, is bound to the first immobilised agent that binds the compoundof formula (I), (II) or (HI), or the other degradation fragment ofbilirubin or biliverdin, or salt thereof. This second agent can belabelled indirectly by contacting with a third antibody specific for theFc region of the second agent, wherein the third agent carries adetectable label.

[0078] Another system which can be used to determine the binding betweenthe compound of formula (I), (II) or (III), or the other degradationfragment of bilirubin or biliverdin, or salt thereof, and the agent is acompetitive binding system. One embodiment of such a system determineswhether the compound of formula (I), (II) or (III), or the otherdegradation fragment of bilirubin or biliverdin, or salt thereof, in thesample is able to inhibit the binding of the agent to a referencecompound which is capable of binding the agent. The reference compoundmay, for example, be a known amount of labelled compound containing afunctional group or groups which the agent recognises. If the compoundof formula (I), (II) or (III), or the other degradation fragment ofbilirubin or biliverdin, or salt thereof, in the sample is able toinhibit the binding the between the agent and reference compound thenthis indicates that such a compound of formula (I), (II) or (III), orother degradation fragment of bilirubin or biliverdin, or salt thereof,is recognised by the agent.

[0079] Examples of detectable labels include enzymes, such as aperoxidase (e.g. of horseradish), phosphatase, radioactive elements,gold (or other colloid metal) or fluorescent labels. Enzyme labels maybe detected using a chemiluminescence or chromogenic based system.

[0080] The invention also includes a dipstick which can be used to carryout the method of the invention. The dipstick comprises a porousmaterial capable of chromatographically transporting a liquid and one ormore of the agents mentioned herein. When the dipstick is contacted withthe sample it draws up liquid from the sample towards a detection regionon the dipstick. Proteins in the sample comprising the polymorphismsmentioned herein are detected by their binding to detection region.

[0081] In one embodiment the liquid is drawn through a region in thedipstick containing the agents mentioned above. The agents bind to thecompound of formula (I), (II) or (III), or the other degradationfragment of bilirubin or biliverdin, or salt thereof, forming anagent/compound complex. This complex is drawn towards the detectionregion which contains a substance (immobilised on the dipstick) thatbinds and thus immobilises the complex in the detection region. Thesubstance is typically a specific binding agent (e.g an antibody) thatbinds either the agent or the compound of the complex. Theagent/compound complex is typically detected in the detection region bythe use of a label which is attached to the agent.

[0082] In another embodiment the compound of formula (I), (II) or (III),or the other degradation fragment of bilirubin or biliverdin, or saltthereof, in the sample is labelled before it is drawn up the dipstick.The labelled compound is then drawn up the dipstick (which has beencontacted with sample) and is detected by binding the antibody (which isbound to the detection region).

[0083] Typically the label used in the dipstick systems described aboveis a visually detectable label which becomes visually detectable (i.e.can be seen with the human eye) when enough agent/compound complexbecomes immobilised in the detection region. A suitable label is a gold(or other colloidal metal) particle or a fluorophore (e.g. fluoroscein).

[0084] In a further aspect the present invention provides a method ofpurifying blood which comprises irradiating it so as to degrade anyphotolabile compounds therein. Such purification of blood may bedesirable in the treatment of, for example, systemic inflammatoryresponse syndrome (SIRS) or other inflammatory disorders, infections,trauma (particularly muscle trauma where haem from myoglobin may beproblematic) or haemolytic conditions.

[0085] In one embodiment the photolabile compounds which are degradedare photolabile degradation fragments of bilirubin or biliverdin.

[0086] The photolabile compounds may be degraded by contacting the bloodwith a blood dialyser which incorporates an irradiator.

[0087] The present invention is further illustrated, merely by way ofexample, with reference to the figures in which:

[0088]FIG. 1 shows the stimulatory effect of the bilirubin oxidativedegradation products (ox-BR) and CSF from SAH patients (CSF-SAH) on therate of oxygen consumption of the porcine carotid artery (FIG. 1A). Thesteady state rate of oxygen consumption (after 90 minutes) by theporcine carotid artery (n=3) in the presence of ox-BR (finalconcentration 1 mg/mL), CSF-SAH (final dilution 1/30) and control (A) issignificantly increased in both cases compared to control (*P≦0.05). Thetime course for the stimulation of oxygen consumption was also similar.Vascular smooth muscle tension development (n=4) is induced by ox-BR(final concentration 1 mg/mL) and CSF-SAH (final dilution 1/30) and canbe abrogated by dobutamine (DOB) pre-treatment of carotid artery rings(B). Results are expressed as mean ±SE.

[0089]FIG. 2 shows the HPLC elution profile of chloroform extract fromcrude oxidised BR analysed on Spherisorb reversed phase column.

[0090]FIG. 3 shows the possible outline pathways for H₂O₂ mediatedformation of Box A, Box B and 4-methyl-3-vinylmaleimide from BR orbiliverdin. In the pathway leading to Box A production: R₁ is —CH₃, R₂is —CH═CH₂, R₃ is —CH₃ and R₄ is —CH₂—CH₂—COOH. In the pathway leadingto Box B formation: R₁ is —CH═CH₂, R₂ is —CH₃, R₃ is —CH₃ and R₄ is—CH₂—CH₂—COOH. In the pathway leading to 4-methyl-3-vinylmaleimide: R ispart of bilirubin molecule.

[0091]FIG. 4 shows the haemorrhage in the brain of a rat following BOXesinjection. FIG. 4a shows a non-BOXes (control) group rat brain and FIG.4b shows a BOXes group rat brain.

[0092]FIG. 5 shows in vivo vasospasm in a rat brain following BOXestreatment. FIG. 5a shows the rat brain with the dura intact and FIG. 5bshows that after BOXes have been dropped onto the surface of the durathere is obvious vasospasm (arrow).

[0093]FIG. 6 shows rat brain slices following topical application ofBOXes. FIG. 6a shows a control rat brain slice and FIG. 6b shows HSP25expression in a rat brain slice after treatment with BOXes.

[0094] The Examples which follow further illustrate the presentinvention with reference to the figures.

EXAMPLES

[0095] Materials and Methods

[0096] Bilirubin Peroxidation Procedure

[0097] Bilirubin (mixed isomers, 100 mg, from Sigma Chemical Co.) wassuspended with stirring in 25 mL of 5 M NaOH over 4 h at roomtemperature. The reaction flask was protected from light by aluminiumfoil. After 4 h the orange suspension of BR had largely dissolved givinga dark green solution. The solution was neutralised using 11 M HCl and20 mL of 30% H₂O₂ was added. The reaction mixture was then incubated inthe dark 48 h, at room temperature, resulting in a transparent yellowsolution with some precipitation. A portion of this crude mixture wasfiltered (pore size 0.2 μm), evaporated to dryness and analysed by ¹HNMR spectroscopy (500 MHz). Another portion of crude mixture was usedfor oxygen consumption measurement. For this examination a portion ofcrude mixture was freeze-dried and re-dissolved in physiological salinesolution (PSS, which is described below) to form a final concentrationof 1 mg/mL of PSS. The remaining reaction mixture was filtered andextracted 10 times with chloroform [reaction mixture: chloroform (5:1)].The solution was dried over sodium sulphate and evaporated in vacuo togive a yellow solid. The latter was also analysed by ¹H NMR spectroscopy(500 MHz).

[0098] HPLC Analyses and Purification

[0099] The chloroform extract was dissolved in 50% (v/v) aqueous andpurified by HPLC. Experiments were conducted so as to exclude light fromsamples. Hypersil reversed-phase CIS (250×7 mm) or Spherisorb ODS (2)reversed-phase columns (250×4.6 mm) were equilibrated with 30% (v/v)aqueous acetonitrile at a flow rate pf 2 mL min⁻¹ and 1 mL min⁻¹,respectively. Elution was performed using 30% to 36% aqueousacetonitrile linear gradient at the same flow rates over 8 min.Fractions were monitored at 320 nm and collected manually. The fractionswere freeze dried and stored at −80° C. for further analyses. Eachfraction was re-dissolved in 50% (v/v) aqueous acetonitrile, re-injectedand re-purified using the same conditions to remove impurities fromother fractions. To investigate the light sensitivity of the compounds,purified fractions were exposed to sunlight for 90 min after dissolving50% (v/v) aqueous and analysed by HLPC.

[0100] Spectroscopy

[0101] UV/Vis spectroscopy was performed in double distilled H₂O usingan Ultrospec 4000 UV/Vis spectrophotometer IR spectra were collected ata 4 cm⁻¹ resolution on a Perkin Elmer FT-IR spectrometer A minimum of256 scans were summed and collected.

[0102] Spectra were recorded at temperature of 300 K on a Bruker AMX500spectrometer (500 MHz) equipped with an inverse-broadband z-gradientprobehead. Heteronuclear multiple-bond correlation (HMBC) spectra wererecorded with gradient selection and without the use of a low-passfilter so that single-bond correlations could be establishedsimultaneously. These were identified by the characteristic ¹J_(CH)doublet structure that is apparent when broadband carbon decoupling isnot applied during collection of each of FID. All ¹³C chemical shiftswere obtained indirectly from correlation peaks in the 2D experiment

[0103] Electrospray Ionisation Mass Spectrometry and Exact MassMeasurements.

[0104] Electrospray ionisation mass spectra were measured on a MicromassBioQ II-ZS triple quadrupole mass spectrometer equipped with anelectrospray interface. Samples (10 μL) were introduced into theelectrospray source via a loop injector. Positive ion spectra were runfrom a solution in water/acetonitrile (1:1 v/v) containing 0.2% (v/v)formic acid at a cone voltage of +30V. Negative ion mass spectra wererun from a solution water/acetonitrile (1:1 v/v) at cone voltages of—10V and —15V, the source temperature was set at 40° C. High-resolutionexact mass spectra were recorded on a Micromass Autospec 5000 OA-Tofmass spectrometer in the chemical ionisation mode with ammonia as thereagent gas.

[0105] Cerebrospinal Fluid (CSF) Collection

[0106] CSF was collected from SAH patients either at the time of surgeryor through the lumbar drain inserted to relieve intracranial pressure.

[0107] Tissue Collection and Measurements of Oxygen Consumption

[0108] Porcine carotid artery were collected from an abattoir within 30minutes of death, rinsed and immersed in PSS at 4° C., and immediatelytransported to the laboratory. Arteries were then trimmed of excessconnective tissue and adventitia to be stored in PSS at 4° C. until use.The PSS was changed every 12 hours and the vessels were kept for up to 4days. The PSS contained the following: 118 mmol/L sodium chloride, 25mmol/L sodium hydrogen carbonate, 5.76 mmol/L potassium chloride, 2.5mmol/L calcium chloride, 1.2 mmol/L magnesium sulphate, 0.5 mmol/Lmonobasic sodium phosphate, and 11.1 mmol/L glucose. The solution wasoxygenated by bubbling with 95% oxygen and 5% carbon dioxide to maintaina pH of 7.4.

[0109] The rate of the oxygen consumption was determined using aHansatech Instruments (Norfolk, United Kingdom) Clark oxygen electrode.Carotid artery rings approximately 0.2 cm long were vigorously rubbed toremove the endothelial cells and were added to the water-jacketedchambers containing PSS at 37° C. The rates of oxygen consumption weremeasured before and after the addition of crude mixture of the BRoxidative degradation products (final concentration 1 mg/mL of PSS).CSF_(SAH) (final dilution 1/30) or 4-methyl-3-vinylmaleimide (purifiedon HPLC, freeze-dried and then re-dissolved in PSS). The results arepresented as μmol O₂ consumed per minute per gram dry weight, assumingthat the solubility of oxygen in PSS at 37° C. is 0.202 μmol/mL or aspercentage increase in oxygen consumption in comparison with oxygenconsumption by carotid artery alone.

[0110] Force Measurements

[0111] Isometric force measurement were performed on porcine carotidartery rings to assess functional parameters during exposure to variouscompounds. The methods used were those used by J F Clark et al., J.Vasc. Res., 1995, 32, 24-30. KCl induced contraction is used as maximalisometric tension generation and results are reported as percentage ofKCl maxima obtained for each carotid.

[0112] Statistical Analysis

[0113] The programme ANOVA was used to statistically evaluate the data,and values were considered to be significantly different if p<0.05.

[0114] Results

[0115] Oxygen Consumption Measurements

[0116] Crude oxidised BR at concentration of 1 mg/mL PSS increasedoxygen consumption of the porcine carotid artery by about 3.6 fold in 90min (FIG. 1). CSF_(SAH) stimulated an increase in the rate of oxygenconsumption in a similar manner as crude oxidised BR (FIG. 1). Highdoses of either crude oxidised BR or CSF_(SAH) from patients with SAHinhibited oxygen consumption by carotid artery or even inhibited thetissue's oxygen consumption. CFS from healthy controls or controlsolutions for crude oxidised BR did not exhibit any activity. Chloroformextracts of crude oxidised BR and 4-methyl-3-vinylmaleimide alsostimulated oxygen consumption. However, it was not possible determinethe concentration of 4-methyl-3-vinylmaleimide used in the experimentsbecause of the small amount of material obtained.

[0117] HPLC Analyses and Purification.

[0118] Following the observation that biological activity wasextractable into chloroform the extracts were analysed and purified byHPLC. FIG. 2 shows the separation pattern for three isolated compounds:Box (Bilirubin oxidised) A, Box B and 4-methyl-3-vinylmaleimide (Box C).Reaction of biliverdin with H₂O₂ also resulted in production of the samecompounds, as judged by their retention times.

[0119] Box A, Box B and 4-methyl-3-vinylmaleimide were light sensitive.When exposed to sunlight for 90 min, as judged by HPLC, the intensity oftheir peaks significantly decreased. ¹H NMR analyses of Box A and Box Bafter sunlight exposure for 90 min also indicated degradation to anumber of products. In contrast, Box A, Box B and4-methyl-3-vinylmaleimide were relatively heat-stable when kept 60° C.for 2 h as judged by HPLC analyses.

[0120] Spectroscopic Analyses

[0121] Box A:4-methyl-5-oxo-3-vinyl-(1,5-dihydroppyrrol-2-ylidene)acetamide (FIG. 3).UV/Vis spectroscopy: λ_(max) 215 nm and 300 nm. IR spectroscopy:3434.0(N—H), 1698.2 (C═O, lactam), 1666.4 (C═O; amide), 1430.5 and1292.7 cm⁻¹. ¹H and ¹³C NMR assignments (500 MHz) were established froma long-range heteronuclear ¹H-¹³C 2D correlation (HMBC) experiment(Table 1). TABLE 1 ¹H NMR ¹³C NMR (100% CD₃CN)^(a) (CD₃ CN/D₂O)^(b) 19.69 — 2 — 147.0 3 — 140.7 4 — 131.5 5 — 173.8 1′ 5.62  99.1 2′ — 169.73-vinyl-CH 6.57 125.3 3-vinyl-CH₂ 5.68 124.6 5.71 4-Me 1.98  9.1 NH₂6.37/5.86 —

[0122] The proton spectrum (in CD₃CN) demonstrated the presence of asingle vinylic CH═CH₂ group plus a sharp singlet consistent with aremote alkene moiety. Also present in this region were two, broadresonances each corresponding to a single proton, which were not presentin later D₂O/acetonitrile spectra. These were suggestive of a primaryamide NH, group in which the two protons were differentiated as a resultof restricted amide bond rotation. A further broad, exchangeableresonance, also corresponding to a single proton, was observed at 9.69ppm indicating the presence of a core pyrrole ring structure. Thesedata, together with the presence of an additional three-proton singletat 1.98 ppm, suggested a pyrrole derived core was intact in thisfragment and that it carried a methyl and vinylic group, and hence thatit resulted from oxidative cleavage site was consistent with molecularmass, and was confirmed from connectivities observed in long-rangeheteronuclear ¹H-¹³C 2D correlation (HMBC) experiments (in 3:7CD₃CN:D₂O, due to limited solubility). All observed correlations wereconsistent with Box A. Notably, the methyl singlet correlated stronglyto a carbonyl at 173.8 ppm whilst the remote alkene proton correlated tothe amide carbonyl at 169.7 ppm. These data also established thelocation of the methyl group adjacent to the carbonyl of the ring. Theassignment of the exocyclic double bond was not possible. Massspectrometry: m/z 179.20 (MN⁺; electroionization); m/z 179.082163 (MH⁺),calc. mass 179.082053 (high-resolution mass spectrometry). Molecularformula: C₉H₁₁N₂O₂.

[0123] Box B:3-methyl-5-oxo-4-vinyl-(1,5-dihydropyrrol-2-ylidene)acetamide (FIG. 4).UV/Vis spectroscopy: λ_(max) 215 nm and 310 nm. IR spectroscopy 3435.7(N—H), 1654.1 (C═O; lactam) and 1647.9 (C═O; amide) cm⁻¹. ¹H and ¹³C NMRassignments (500 MHz) were established from a long-range heteronuclear¹H-¹³C 2 D correlation (HMBC) experiment (Table 2). TABLE 2 ¹H NMR ¹³CNMR (100% CD₃CN)² (CD₃CN/D₂O)^(b) 1 9.49 — 2 — 147.9 3 — 142.7 4 — 129.25 — 173.0 1′ 5.55 99.0 2′ — 170.2 4-vinyl-CH 6.62 125.7 4-vinyl-CH₂5.52, 6.32 122.4 3-Me 2.07 9.1 NH₂ 6.37/5.86 —

[0124] The proton spectra demonstrated the same principle features asfor Box A, with the most significant being in the shift dispersion ofthe vinylic protons, HMBC data (in 3:7 CD₃CN:D₂O) confirmed the presenceof the same core structure but indicated the relative position of themethyl and vinyl groups were switched. The CH proton of the vinyl groupnow correlated strongly to the carbonyl of the pyrrole ring at 173.0ppm. This isomeric fragment would therefore derive from oxidativecleavage from the other end of the BR molecule to that which producedBox A. The assignment of the exocyclic double bond stereochemistry wasnot possible. Mass spectrometry: m/z 179 (MH⁻, electroionization); m/z179.082021 (MH⁻), calc. mass 179.082053 (high-resolution massspectrometry). Molecular formula: C₉H₁₁N₂O₂.

[0125] The shift patterns observed for the methyl and vinyl protons inBox A and Box B bear a striking similarity with those of the tri-pyrrolefragments: biotripyrrin-a and biotripyrrin-b (FIG. 3) isolated byYamaguchi et al (Yamaguchi 94). In both cases, the vinylic protons inparticular display characteristic behaviour, with the geminal pairappearing essentially coincident when they sit on the opposite side ofthe pyrrole core to the pyrrole carbonyl, but displaying significantdispersion when adjacent to this carbonyl group. These similaritiesfurther support the assigned relative location of-methyl and vinylgroups in Box A and Box B.

[0126] 4-Methyl-3-vinylmaleimide: IR spectroscopy: 3436.2 (N—H), 2103.9,1773.7, 1713.0, 1639.6 cm⁻¹. ¹H and ¹³C NMR assignments (500 MHz) wereestablished from a long-range heteronuclear ¹H-¹³C 2D correlation (HMBC)experiment (Table 3). TABLE 3 ¹H NMR^(a) ¹³C NMR^(b) 1 7.19 — 2 — 170.23 — 134.3 4 — 136.7 5 — 171.4 3-vinyl-CH 6.55 124.2 3-vinyl-CH₂ 6.40,5.73 125.9 4-Me 2.07 8.3

[0127] 4-Methyl-3-vinylmaleimide was soluble in chloroform and affordeda simple ¹H spectrum, demonstrating the presence of a vinyl group (inwhich the geminal pair were well dispersed), a methyl group and a broadresonance at 7.19 ppm. HMBC spectra demonstrated correlations from thevinyl CH proton and from the methyl group to different carbonyl centres,and readily identified the fragment as having the structure of4-methyl-3-vinylmaleimide. The dispersion of the vinylic protons isagain consistent with these being adjacent to a carbonyl group withinthe pyrrole core. Negative ion mass spectrometry m/z 135.9. ¹H NMR (500MHz) and mass spectrometry data were consistent with those reported for4-methyl-3-vinylmaleimide.

[0128] BR was shown to react with hydrogen peroxide, using a proceduredesigned to mimic severe oxidative stress, to give various fragmentationproducts. The crude oxidised BR solution stimulated oxygen consumptionof vascular smooth muscle from the porcine carotid artery in the absenceof endothelial layer. CSF_(SAH) also stimulated oxygen consumption invascular smooth muscle. Comparison of CSF_(SAH) with the BR oxidativedegradation products in the oxygen consumption assays revealed that theybehaved in a similar manner (FIG. 1). CSF_(SAH) and the BR oxidativedegradation products induced stimulation of oxygen consumption followingaddition to the chamber with the artery. In addition, high doses of bothCSF_(SAH) and the BR oxidative degradation products inhibited the rateof oxygen consumption or even killed vascular smooth muscle. Isometricforce measurement experiments confirmed that the change in metabolismmirrored the changes caused by CSF_(SAH) and the BR oxidation products.

[0129] The bioactive compounds from both CSF_(SAH) and oxidised BR wereboth extractable into chloroform. Three photo-labile compounds wereisolated by HPLC:4-methyl-5-oxo-3-vinyl-(1,5-dihydropyrrol-2-ylidene)acetamide (Box A)and 4-methyl-3-vinylmaleimide and3-methyl-5-oxo-4-vinyl-(1,5-dihydropyrrol-2-ylidene)acetamide (Box B).Along with Box A and Box B the monopyrrole derivative4-methyl-3-vinylmaleimide was also isolated. 4-Methyl-3-vinylmaleimideis known to be formed during photooxidation of biliverdin, as well as inthe reaction of H₂O₂ with ferri-protoporphyrin IX or by chromic acidwith BR. The reaction mixture was protected from light, therefore it isassumed that 4-methyl-3-vinylmaleimide is formed in the reaction of H₂O₂with BR or biliverdin. Nevertheless, traces of 4-methyl-3-vinylmaleimidemay be produced by incidental light exposure. Although4-methyl-3-vinylmaleimide has not been detected directly in vivo, itshydrolysis has been found in jaundiced neonates undergoing phototherapy.

[0130] Box A and Box B cannot be formed using the mechanism leading tobiotripyrrins a and b, since formation of official Box A and Box Brequires nitrogen from pyrrole rings. Studies on singlet oxygen mediatedphotolytic degradation of BR have indicated a mechanism involvingelectron transfer of excited state BR with ground state dioxygen. Anumber of products including biliverdin, 4-methyl-3-vinylmaleimide andsimple aliphatic acids have been identified. Whether or not biliverdinis an intermediate in some or all of the fragmentation products isunclear, but they, like BR are not light stable.

[0131] A possible mechanism of Box A, Box B and4-methyl-3-vinylmaleimide during photooxidation is proposed in FIG. 3. ABR-dioxetane has been proposed as an intermediate to4-methyl-3-vinylmaleimide during photooxidation. However, theintermediates of such species seems less likely in H₂O₂ mediatedoxidation. Attack of H₂O₂ at C-4 or C-16 to give the peroxide shown inFIG. 3, followed by Crigee rearrangement and hydrolysis to give4-methyl-3-vinylmaleimide may be more likely. The mechanism of initialattack of peroxide is uncertain, with formation of a (radical) cationderivative of BR or biliverdin as a possibility. Similarity, formationof peroxides at C-6 or C-14 followed by rearrangement and hydrolysis canlead to Box A and Box B.

[0132] Like 4-methyl-3-vinylmaleimide, which has been shown to reactwith thiols, including glutathione, Box A and Box B are alkylatingagents (Michael acceptors). It is possible they exert biologicalactivity leading to vasospasm via an alkylation process, e.g. affectingcellular phosphatase/kinase systems.

[0133] BR oxidative degradation products exhibits biological activityand may play a role in pathogenesis of arterial vasospasm followinghaemorrhage. Oxidation of BR leads to production of BR-derived fragmentswhich present in CSF_(SAH) from SAH patients.

[0134] Evidence for the Presence of Box A in Human Cerebral Spinal Fluid(CSF) Method

[0135] Double deionised water (100 ml) was added to 45 ml dry volume offreeze dried CSF and stirred in the dark for 45 mins. Whilst stirringcontinuously in the dark, urea (20 g) was then added to the (partially)reconstituted CSF to give an approximate concentration of 3M urea. Afteraddition of urea, the solution was stirred for a further 5 mins to givea fully reconstituted solution of CSF with no solid extract. 500 ml ofchloroform was then added to this solution whilst stirring in the dark.The solution was then stirred for one hour then centrifuged at 2800 rpmfor 5 mins. The chloroform layer was then collected and dried overanhydrous magnesium sulphate in the dark. The magnesium sulphate wasremoved by filtration in the dark and the chloroform removed by rotaryevaporation in vacuo in the dark. The resultant solid extract wasredissolved in 250 μl of 1:1 acetonitrile:deionised water. The resultantsolution was then configured at 13000 rpm for 15 mins.

[0136] The supernatant was then analysed by HPLC equipped with aphotodiode array detector. The experimental conditions were as follows:100 μl of the 1:1 acetonitrile:deionised water solution was analysed byHPLC using a Phenomenex Phenosphere 5 micron ODS2 80A 250 mm×4.6 mmcolumn. The following gradient was run with a flow rate of 1 ml aminute. % Buffer A 10 mM Ammonium % Buffer B bicarbonate in 50% 10 mMAmmonium Acetonitrile/50% bicarbonate in deionised Time in minutesdeionised water water 0-5 50 50  5-30 50-100 50-0 (linear gradient)(linear gradient) 30-35 100   0 35-45 50 50

[0137] At a retention time of ca. 5 min 20 secs a compound with UVprofile with absorbance maxima at 212 and 297 nm was observed. Theprofile was the same as that of a reference sample Box A (preparedsynthetically). The presence of Box A in CSF was further confirmed byanalysis of CSF samples doped with reference Box A.

[0138] Mortality Study Using Bilirubin Oxidation Products (BOXes)

[0139] A mortality study was performed in which lysed autologus bloodwas injected into the cisterna magna of anesthetized rats. In one groupof rats the lysed autologous blood was supplemented with a standardizedamount of BOXes and in the other group BOXes were absent (with constantvolumes of solutions (50 μL) injected in both groups). The BOXes groupcontained 7 rats and the non-BOXes (control) group contained 12 rats.The BOXes were prepared as previously described and the same doseadministered as used in the oxygen consumption measurements describedabove.

[0140] In the BOXes group, 5 of the 7 rats died within 24 hours whereasin the non-BOXes group none of the 12 rats died. This is a significantincrease in mortality (P≦0.001) as determined with the Fishers ExactTest.

[0141] The cause of death was believed to be intense vasoconstrictioncausing ischemic damage as well as visible signs of haemorrhage. Thehaemorrhage is likely to be due to venous hypertension and subarachnoidhaemorrhage (see FIG. 4b showing the haemorrhage in the brain of a ratfollowing BOXes injection).

[0142] Vasospasm in Rats Caused by BOXes

[0143] A lower dose of BOXes than that injected into the cisterna magnain the mortality study was dropped onto the surface of a rat brain.Typically, 200 μL of BOXes was dropped on to the surface of the brainbut a significant proportion of the solution simply washed over thesurface of the brain's dura matter. A vasospasm response was observed inless than 10 minutes (see FIG. 5b).

[0144] Induction of Heat Shock Proteins by BOXes

[0145] The brains from the animals used in the vasospasm study wereprobed for heat shock proteins (HSP); HSP 32, HSP 70 and HSP 25. All ofthese heat shock proteins were found to be induced in the underlyingcortex where the BOXes were applied and where the vasospasm wasobserved. HSP 25 expression localized to the blood vessels in theseregions was also observed (see FIG. 6b).

[0146] The induction of HSPs was not evident in the control animals(bilirubin or saline). The induction of HSPs was localized to the areawhere the BOXes were applied.

1. A pharmaceutical composition comprising a compound of formula (I)

wherein X is an electron withdrawing group, Y¹ is hydrogen, alkyl,alkenyl, alkynyl, aryl, heterocyclyl, —SO₂R⁴, —CO₂R⁴, —CONHR⁴ or —COR⁴,and each of R¹, R² and R⁴, which may be the same or different, ishydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl orheterocyclyl, or a compound of formula (II)

wherein each of Y² and Y³, which may be the same or different, ishydrogen, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, —SO₂R⁹, —CO₂R⁹,—CONHR⁹ or —COR⁹, Z is hydrogen, alkyl, cycloalkyl, alkenyl,cycloalkenyl, alkynyl, aryl, heterocyclyl,—CH═C(NHR¹⁰)CH((CH₂)_(m)CO₂R¹¹)(C═O)CH₃ or—CH₂(C═O)CH((CH₂)_(m)CO₂R¹¹)(C═O)CH₃, R⁸ is —(CH₂)_(n)CO₂R¹², each of R⁵to R⁷ and R⁹ to R¹², which may be the same or different, is hydrogen,alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl or heterocyclyl,and each of m and n, which may be the same or different, is 1 to 6, or acompound of formula (III)

wherein each of Y⁴ to Y⁶, which may be the same or different, ishydrogen, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, —SO₂R¹⁹, —CO₂R¹⁹,—CONHR¹⁹ or —COR¹⁹, each of R¹⁶ and R¹⁷, which may be the same ordifferent, is —(CH₂)_(p)CO₂R²⁰, each of R¹³ to R¹⁵ and R¹⁸ to R²⁰, whichmay be the same or different, is hydrogen, alkyl cycloalkyl, alkenyl,cycloalkenyl, alkynyl, aryl or heterocyclyl, and p is 1 to 6, or otherphotolabile degradation product of bilirubin or biliverdin or derivativeof a photolabile degradation product of bilirubin or biliverdin, or apharmaceutically acceptable salt thereof, together with apharmaceutically acceptable carrier or diluent.
 2. A compositionaccording to claim 1 wherein in formula (I) X is ═O, ═CH(C═O)R³,═CH(C═O)OR³ or ═CH(C═O)NHR³ and R³ is hydrogen, alkyl, cycloalkyl,alkenyl, cycloalkenyl, alkynyl, aryl or heterocyclyl.
 3. A compositionaccording to claim 1 or claim 2, wherein in formula (I) X is ═O orCH(C═O)NHR³, each of R¹ and R², which may be the same or different, ishydrogen, alkyl or alkenyl and R³ is hydrogen or alkyl, or in formula(II) Z is —CH═C(NHR¹⁰)CH((CH₂)_(m)CO₂R¹¹)(C═O)CH₃ or—CH₂(C═O)CH((CH₂)_(m)CO₂R¹¹)(C═O)CH₃, each of R⁵ to R⁷ is hydrogen,alkyl or alkenyl, R⁸ is —(CH₂)₂CO₂R¹², each of R¹⁰ to R¹² is hydrogen oralkyl and m is 1 to 6, or in formula (III) each of R¹³ to R¹⁵ and R¹⁸are hydrogen, alkyl or alkenyl each of R¹⁶ and R¹⁷ is —(CH₂)₂CO₂R²⁰ andR²⁰ is hydrogen or alkyl.
 4. A composition according to any one of thepreceding claims wherein in formula (I) when X is ═CH(C═O)NH₂, Y¹ ishydrogen, and one of R¹ and R² is hydrogen or alkyl and the other isalkenyl, or when X is ═O, Y¹ is hydrogen, R¹ is alkenyl and R² ishydrogen or alkyl, or in formula (II) Y² and Y³ are hydrogen, Z is—CH═C(NHR¹⁰)CH((CH₂)_(m)CO₂R¹¹)(C═O)CH₃ or—CH₂(C═O)CH((CH₂)_(m)CO₂R¹¹)(C═O)CH₃, one of R⁵ and R⁶ is hydrogen oralkyl and the other is alkenyl, R⁷ is alkyl, R⁸ is (CH₂)₂CO₂H, each ofR¹⁰ and R¹¹ is hydrogen or alkyl and m is 1 to 4, or in formula (III)each of Y⁴ to Y⁶ is hydrogen, one of R¹³ and R¹⁴ is hydrogen or alkyland the other is alkenyl, each of R¹⁵ and R¹⁸ is hydrogen or alkyl andeach of R¹⁶ and R¹⁷ is —(CH₂)₂CO₂H.
 5. A composition according to anyone of the preceding claims, wherein in formula (I) when X is═CH(C═O)NH₂, Y¹ is hydrogen, and one of R¹ and R² is methyl and theother is —CH═CH₂, or when X is ═O, Y¹ is hydrogen, R¹ is —CH═CH₂ and R²is methyl, or in formula (II) each of Y² and Y³ is hydrogen, Z is—CH═C(NH₂)CH((CH₂)₂CO₂H)(C═O)CH₃ or —CH₂(C═O)CH((CH₂)₂CO₂H)(C═O)CH₃, oneof R⁵ and R⁶ is methyl and the other is —CH═CH₂, R⁷ is methyl and R⁸ is—(CH₂)₂CO₂H, or in formula (III) each of Y⁴ to Y⁶ is hydrogen, one ofR¹³ and R¹⁴ is methyl and the other is —CH═CH₂, each of R¹⁵ and R¹⁸ ismethyl and each of R¹⁶ and R¹⁷ is —(CH₂)₂CO₂H.
 6. A compound of formula(I), (H) or (III) as defined in any one of claims 1 to 5, or otherdegradation fragment of bilirubin or biliverdin or derivative of adegradation fragment of bilirubin or biliverdin, or a salt thereof,excluding a compound of formula (I) wherein X is ═O, Y¹ is hydrogen, R¹is —CH═CH₂ and R² is methyl, or a compound of formula (III) wherein eachof Y⁴ to Y⁶ is hydrogen, one of R¹³ or R¹⁴ is methyl and the other is—CH═CH₂, each of R¹⁵ and R¹⁸ is methyl and each of R¹⁶ and R¹⁷ is—(CH₂)₂CO₂H.
 7. A diagnostic composition comprising a compound offormula (I) as defined in claim 1 wherein when X is ═CH(C═O)NH₂, Y¹ ishydrogen, and one of R¹ and R² is methyl and the other is —CH═CH₂, orwhen X is ═O, Y¹ is hydrogen, R¹ is —CH═CH₂ and R² is methyl, or acompound of formula (II) as defined in claim 1 wherein each of Y² and Y³is hydrogen, Z is —CH₂(C═O)CH((CH₂)₂CO₂H)(C═O)CH₃, one of R⁵ and R⁶ ismethyl and the other is —CH═CH₂, R⁷ is methyl, R⁸ is (CH₂)₂CO₂H, or acompound of formula (III) wherein one of R¹³ and R¹⁴ is methyl and theother is —CH═CH₂, each of R¹⁵ and R¹⁸ is methyl and each of R¹⁶ and R¹⁷is —(CH₂)₂CO₂H, or other degradation fragment of bilirubin orbiliverdin, or a salt thereof, and a diluent or carrier.
 8. Acomposition according to claim 7 wherein the compound is photolabile. 9.A method for diagnosing vasospasm or vasoconstriction in a hostcomprising determining the presence or absence of a compound of formula(I), (II) or (III), or other degradation fragment of bilirubin orbiliverdin, or salt thereof, as defined in claim 7 or claim 8, whereinthe presence of the compound of formula (I), (II) or (III), or thedegradation fragment of bilirubin or biliverdin, indicates that the hosthas vasospasm or vasoconstriction.
 10. A method according to claim 9,which method comprises (c) contacting a sample from the host with anagent that binds to the compound of formula (I), (II) or (III), or theother degradation fragment of bilirubin or biliverdin, or salt thereof,and (d) detecting whether the agent binds to components in the sample,thereby determining the presence or absence of the compound of formula(I), (II) or (III), or the other degradation fragment of bilirubin orbiliverdin, or salt thereof.
 11. A method according to claim 10 whereinthe agent is an antibody which is specific for the compound of formula(I), (II) or (III), or the other degradation fragment of bilirubin orbiliverdin, or salt thereof.
 12. A method according to claim 11 whereinthe antibody is labelled.
 13. A method of purifying blood whichcomprises irradiating it so as to degrade any photolabile compoundstherein.
 14. A method according to claim 13 wherein the photolabilecompounds are photolabile degradation fragments of bilirubin orbiliverdin.
 15. A blood dialyser which incorporates an irradiator.
 16. Acompound of formula (I), (II) or (III), or other photolabile degradationfragment of bilirubin or biliverdin or derivative of a degradationfragment of bilirubin or biliverdin, or a pharmaceutically acceptablesalt thereof, as defined in any one of claims 1 to 5 for use in a methodof treatment of the human or animal body.
 17. Use of a compound offormula (I), (II) or (III), or other photolabile degradation fragment ofbilirubin or biliverdin or derivative of a degradation fragment ofbilirubin or biliverdin, or a pharmaceutically acceptable salt thereof,as defined in any one of claims 1 to 5 in the manufacture of amedicament for use in treating or inducing vasospasm orvasoconstriction.